ABSTRACT ? Project 3: Chemical probe discovery for PAX3-FOXO1 The majority of pediatric alveolar rhabdomyosarcoma (ARMS) cases are characterized by a chromosomal translocation encoding the PAX3-FOXO1 fusion oncoprotein, a transcription factor correlated with poor patient outcomes. The overarching goal of this FusOnc2 Center is to advance the therapeutic tractability of the PAX3- FOXO1 fusion protein in ARMS by comprehensively identifying the druggable co-regulators, modulators, and intrinsic activities of PAX3-FOXO1. Transcription factor fusion proteins such as PAX3-FOXO1 have been validated through genetic approaches as key therapeutic targets, since they may represent tumor-specific Achilles? heels. Unfortunately, to date there are no small molecules that can modulate the function of PAX3- FOXO1. Hence, novel approaches are needed to develop chemical probes or drugs that can target this fusion oncoprotein. Similar to other fusion transcription factors, PAX3-FOXO1 is intrinsically disordered and lacks the traditional small-molecule binding pockets observed in historically tractable targets, complicating structure-driven design or small-molecule screening efforts to identify drug candidates. This Project?s objective is to develop chemical probes for PAX3-FOXO1 using a novel emerging strategy developed within the Koehler Laboratory against other transcription factors and recalcitrant targets. The strategy involves the use of high-throughput and unbiased binding assays involving small-molecule microarrays (SMMs) screened with purified, full-length transcription factor or transcription factor residing in cell lysates. We will exploit this novel technology to develop chemical probes against PAX3-FOXO1 as the first step in drugging this protein through the following four Specific Aims: 1) Execute binding assays for PAX3-FOXO1 using SMMs containing >55,000 small molecules; 2) Perform phenotypic and biophysical characterization of putative PAX3-FOXO1 binders; 3) Synthetically optimize chemical probes and evaluate target engagement in cells; 4) Conduct mechanistic studies of chemical probes in cellular and murine models, in collaboration with the Validation Core. This Project will establish gross structure- activity relationships for 1-3 compounds and use principles of medicinal chemistry to develop new analogs with improved potency in cellular assays and physicochemical characteristics and will synthesize proteolysis targeting chimeras (PROTACs) in an effort to explore targeted degradation of the PAX3-FOXO1 fusion protein. While the aims of this multi-disciplinary proposal are ambitious, the Project will be enabled by the cumulative experience of the team, as well as the extensive capabilities of our FusOnC2 Center and its Projects and Cores. Our approach will lead to high-quality probes of PAX3-FOXO1 function that may clarify the role of the fusion as a direct therapeutic target for ARMS. Through these efforts we also expect to define general strategies for targeting other recalcitrant oncogenic fusion proteins with small molecules.